Inhibitors of placental growth factor for the treatment of pathological angiogenesis, pathological arteriogenesis, inflammation, tumor formation and/or vascular leakage

ABSTRACT

The present invention relates to the field of pathological angiogenesis and arteriogenesis and, in particular, to a stress-induced phenotype in a transgenic mouse (PIGF −/− ) that does not produce Placental Growth Factor (PIGF) and that demonstrates an impaired vascular endothelial growth factor (VEGF)-dependent response. PIGF deficiency has a negative influence on diverse pathological processes of angiogenesis, arteriogenesis and vascular leakage comprising ischemic retinopathy, tumor formation, pulmonary hypertension, vascular leakage (edema formation) and inflammatory disorders. The invention thus relates to molecules that can inhibit the binding of PIGF to its receptor (VEGFR-1), such as monoclonal antibodies and tetrameric peptides, and to the use of these molecules to treat the above-mentioned pathological processes.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional of application Ser. No.10/291,979, filed Nov. 11, 2002 (allowed, which published as US2003-0180286 A1 on Sep. 25, 2003), which is a continuation ofPCT/EP01/05478, filed May 10, 2001 (which designated the U.S. and waspublished in English as WO 01/85796 A2), which claims benefit of EP00201714.3, filed May 12, 2000, the entire contents of each of which isincorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to the field of pathological angiogenesis andarteriogenesis. In particular, the invention describes a stress-inducedphenotype in a transgenic mouse (PIGF−) that does not produce PlacentalGrowth Factor (PIGF) and that demonstrates an impaired vascularendothelial growth factor (VEGF)-dependent response. It is revealed thatPIGF deficiency has a negative influence on diverse pathologicalprocesses of angiogenesis, arteriogenesis and vascular leakagecomprising ischemic retinopathy, tumor formation, pulmonaryhypertension, vascular leakage (edema formation) and inflammatorydisorders. The invention thus relates to molecules that can inhibit thebinding of PIGF to its receptor (VEGFR-1), such as monoclonal antibodiesand tetrameric peptides. The invention further relates to the use ofthese molecules to treat the above-mentioned pathological processes.

BACKGROUND OF THE INVENTION

Abnormal blood vessel formation contributes to the pathogenesis ofnumerous diseases with high morbidity and mortality. Elucidation of themechanisms underlying vascular growth might allow the development oftherapeutic strategies to stimulate vascular growth in ischemic tissuesor to suppress their formation in tumors. Recent gene targeting studiesin embryos have identified some of the mechanisms involved in theinitial formation of endothelial channels (angiogenesis) and theirsubsequent maturation by coverage with smooth muscle cells(arteriogenesis). Evidence is emerging that distinct molecularmechanisms may mediate growth of blood vessels during pathologicalconditions, but the molecular players remain largely undetermined.

VEGF has been implicated in development and pathological growth of thevasculature (N. Ferrara et al., 1999, Curr. Top. Microbiol. Immunol.237, 1-30). Deficiency of a single VEGF allele causes fatal vasculardefects (P. Carmeliet et al., 1996, Nature 380, 435-439; and N. Ferraraet al., 1996, Nature 380, 439-442), whereas suppression of VEGF in theneonate or expression of a single VEGF 120 isoform results in impairedvascular growth (H. P. Gerber et al., 1999, Development 126, 1149-1159;and P. Carmeliet et al., 1999, Nat. Med. 5, 495-502). In the adult, VEGFaffects vascular growth during reproduction, wound healing, andmalignant and inflammatory disorders (N. Ferrara et al., 1999, Curr.Top. Microbiol. Immunol. 237, 1-30). VEGF is currently being tested fortherapeutic angiogenesis in the ischemic heart and limb, but initialclinical trials have resulted in both promising and disappointingresults (J. M. Isner et al., 1999, J. Clin. Invest. 103, 1231-1236). Anoutstanding question is whether VEGF is able to stimulate the maturationof vessels with a smooth muscle coat (arteriogenesis). Naked endothelialchannels remain vulnerable to traumatic insults, regress during changesin oxygen, and lack vasomotor control to accommodate changes in tissueperfusion (L. E. Benjamin et al., 1998, Development 125, 1591-1598). Insome diseases such as pulmonary hypertension, excess arteriogenesis isan undesired and poorly controllable phenomenon. In pulmonaryhypertension, remodeling of the pulmonary vasculature occurs becausevascular smooth muscle cells proliferate and migrate distally around theterminal arterioles, increasing thereby the pulmonary vascularresistance. Another aspect of VEGF is that this molecule affects thepermeability and growth of adult quiescent vessels. In normal humanserum, no detectable levels of VEGF are present, but under pathologicalconditions, such as cancer and inflammatory disorders, VEGF is highlyup-regulated and mediates the formation of undesired edema. Edemaformation is also an important clinical problem associated with severaltumors leading to ascites in peritoneal tumors, pleuritis in lung cancerand cerebral edema in brain tumors (possibly leading to fatalintracranial hypertension) and often facilitates metastasis of tumors.Vascular congestion and edema are important pathogenic mechanisms inasthma, brain infarct expansion after stroke, peritoneal sclerosis afterdialysis or abdominal interventions, etc. Other VEGF homologues havebeen identified, but their role in angiogenesis and arteriogenesisremains unclear.

One interesting homologue of VEGF is Placental Growth Factor (PIGF) butits role in vascular growth and pathogenesis has been poorly studied (M.G. Persico et al., 1999, Curr. Top. Microbiol. Immunol. 237, 31-40).U.S. Pat. No. 5,919,899 describes PIGF and its use in the treatment ofinflammatory disorders, wounds and ulcers. Donnini et al. (J. Pathol.189, 66, 1999) have observed a correlation between up-regulation of PIGFand human meningiomas but it is clear that there is no indicationwhatsoever that PIGF has a role in tumor formation. The role of PIGF inedema was studied by Monsky et al. (Cancer Res. 59, 4129, 1999), but noin vivo role for PIGF in edema formation during pathological processescould be found in several mouse and human tumors.

Inhibitors for PIGF are not known in the art except for a goatpolyclonal antibody against human PIGF (R&D Pharmaceuticals, Abingdon,UK) and a chicken polyclonal antibody (Gassmann et al., 1990, Faseb J.4, 2528). Those antibodies are used for western blotting, histochemistryand immunoprecipitation studies. The role of the PIGF receptor(=VEGFR-1) for endothelial cell biology has also remained enigmatic (A.Sawano et al., 1996, Cell Growth Differ. 7, 213-221 and M. Clauss etal., 1996, J. Biol. Chem. 271, 17629-17634). Gene-targeting studiesyielded conflicting results on the role of VEGFR-1, either as a possiblesignaling receptor (suggested by the vascular defects inVEGFR-1-deficient embryos (G. H. Fong et al., 1999, Development 126,3015-3025)) or as an inert binding site, a “sink,” for VEGF, regulatingavailability of VEGF for the angiogenic VEGFR-2 (suggested by the normalvascular development in mice expressing a truncated VEGFR-1, lacking thetyrosine kinase domain (S. Hiratsuka et al., 1998, Proc. Natl. Acad.Sci. U.S.A. 95, 9349-9354)).

The present invention relates to the surprising finding that PIGF is aspecific modulator of VEGF during a variety of pathological conditions,such as ischemic retinopathy, tumorigenesis, inflammatory disorders,wound healing, edema and pulmonary hypertension. This finding hasimplications for the inhibition of vascular leakage (edema formation),inflammatory disorders, tumor formation, pathological angiogenesis andthe prevention of pulmonary hypertension that occurs during pathologicalarteriogenesis.

DISCLOSURE OF THE INVENTION

The present invention aims at providing research tools and therapeuticsfor patients suffering from pathological angiogenesis, pathologicalarteriogenesis and edema formation. In particular, the invention aims atproviding molecules, such as antibodies, small molecules, tetramericpeptides, ribozymes, antisense nucleic acids, receptor antagonists orsoluble receptors that can block the activity and/or synthesis of PIGFor antagonize the VEGFR-1 activity or can inhibit the signaltransduction from the VEGFR-1 to VEGFR-2. The invention further aims atusing these molecules for the treatment and/or the prevention of, butnot limited to, pulmonary hypertension, cancer, edema, ischemicretinopathy and inflammatory disorders. The present invention also aimsat providing a method to screen for molecules that bind on VEGFR-1 orPIGF.

In other words, the present invention aims at providing therapeutics ora medicament that can be used for the treatment of pulmonaryhypertension, tumor formation, edema, ischemic retinopathy orinflammatory disorders.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: Role of PIGF in pathological vascular growth.

Panel A: PIGF—constitutively produced by adult quiescent endothelialcells (EC)—is not essential for maintenance of the adult quiescentvasculature, presumably because it is ineffective in the presence ofminimal VEGF expression. When expression of VEGF is up-regulated duringischemia, inflammation (macrophages: Mφ or malignancy (tumor cells),PIGF amplifies the response of endothelial and smooth muscle cells (SMC)to VEGF, resulting in enhanced angiogenesis, vascular permeability andarteriogenesis. PIGF can act in an autocrine manner on endothelial,smooth muscle and inflammatory cells, but is also produced by nearbytumor cells, ischemic cardiomyocytes, etc. Panel B: In the absence ofPIGF, vessels are normally formed during development, but respond lessto VEGF during pathological conditions.

DETAILED DESCRIPTION OF THE INVENTION

In previous studies, the PIGF gene was inactivated in the mouse genomevia homologous recombination in embryonic stem (ES) cells (P. Carmeliet,2000, J. Pathol. 190, 387-405; P. Carmeliet, 1999, Curr. Interv.Cardiol. Reports 1, 322-335; and P. Carmeliet and D. Collen, 1999, Curr.Top. Microbiol. Immunol. 237, 133-158). PIGF-deficient (PIGF^(−/−)) miceare viable and fertile, and did not exhibit spontaneous vasculardefects. In the present invention, it is shown that growth ofendothelial channels (angiogenesis), vascular maturation by smoothmuscle cells (arteriogenesis) and vascular permeability aresignificantly impaired in adult PIGF^(−/−) mice during a variety ofconditions where pathological angiogenesis and edema formation occurs.The latter conditions comprise ischemic retinopathy, tumor formation,pulmonary hypertension, edema and inflammation also known to involveVEGF. In another aspect of the invention, it is shown that the role ofPIGF is not only restricted to the formation of immature capillaries,but also includes the maturation/stabilization of newly formed vesselsvia stimulating their coverage with smooth muscle cells(arteriogenesis), a therapeutic prerequisite for functional andsustainable angiogenesis, but an undesired effect of pathologicalarteriogenesis as in the case of pulmonary hypertension.

Thus, in one embodiment, the present invention relates to molecules thatcomprise a region that can specifically bind to placental growth factoror to vascular endothelial growth factor receptor-1; these molecules cansuppress or prevent placental growth factor-induced pathologicalangiogenesis, vascular leakage (edema), pulmonary hypertension, tumorformation and/or inflammatory disorders. With “suppression,” it isunderstood that suppression can occur for at least 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or even 100%. More specifically, the inventionrelates to molecules that can be used to neutralize the activity of PIGFby interfering with its synthesis, translation, dimerization,receptor-binding and/or receptor-binding-mediated signal transduction.

By “molecules,” it is meant peptides, tetrameric peptides, proteins,organic molecules, mutants of the VEGFR-1, soluble receptors of VEGFR-1and any fragment or homologue thereof having the same neutralizingeffect as stated above. Also, the invention is directed to antagonistsof PIGF such as anti-PIGF antibodies and functional fragments derivedthereof, antisense RNA and DNA molecules and ribozymes that function toinhibit the translation of PIGF, all capable of interfering/orinhibiting the VEGFR-1 signal transduction.

By “synthesis,” it is meant transcription of PIGF. Small molecules canbind on the promoter region of PIGF and inhibit binding of atranscription factor or the molecules can bind the transcription factorand inhibit binding to the PIGF promoter.

By “PIGF” is also meant its isoforms, which occur as a result ofalternative splicing, and allelic variants thereof. As a result ofalternative splicing, three PIGF RNAs encoding monomeric human PIGF-1,PIGF-2 and PIGF-3 isoform precursors containing 149, 179 and 219 aminoacid residues, respectively, have been described. In normal mousetissues, only one mouse PIGF mRNA encoding the equivalent of humanPIGF-2 has been identified.

In a specific embodiment, the invention provides a murine monoclonalantibody against PIGF. In another specific embodiment, the murinemonoclonal antibody is MabPL5D11. This monoclonal antibody is availablein the Department of Transgene Technology and Gene Therapy, UZGasthuisberg, Herestraat 49, B-3000 Leuven.

The terms “antibody” or “antibodies” relate to an antibody characterizedas being specifically directed against PIGF or VEGFR-1 or any functionalderivative thereof, with the antibodies being preferably monoclonalantibodies, or an antigen-binding fragment thereof, of the F(ab′)₂,F(ab) or single chain Fv type, or any type of recombinant antibodyderived thereof. These antibodies of the invention, including specificpolyclonal antisera prepared against PIGF or VEGFR-1 or any functionalderivative thereof, have no cross-reactivity to other proteins. Themonoclonal antibodies of the invention can, for instance, be produced byany hybridoma liable to be formed according to classical methods fromsplenic cells of an animal, particularly of a mouse or rat immunizedagainst PIGF or VEGFR-1 or any functional derivative thereof, and ofcells of a myeloma cell line, and to be selected by the ability of thehybridoma to produce the monoclonal antibodies recognizing PIGF orVEGFR-1 or any functional derivative thereof that have been initiallyused for the immunization of the animals.

The monoclonal antibodies according to this embodiment of the inventionmay be humanized versions of the mouse monoclonal antibodies made bymeans of recombinant DNA technology, departing from the mouse and/orhuman genomic DNA sequences coding for H and L chains or from cDNAclones coding for H and L chains.

Alternatively, the monoclonal antibodies according to this embodiment ofthe invention may be human monoclonal antibodies. Such human monoclonalantibodies are prepared, for instance, by means of human peripheralblood lymphocytes (PBL) repopulation of severe combined immunedeficiency (SLID) mice as described in PCT/EP 99/03605 or by usingtransgenic non-human animals capable of producing human antibodies asdescribed in U.S. Pat. No. 5,545,806. Also, fragments derived from thesemonoclonal antibodies such as Fab, F(ab)′2 and ssFv (“single chainvariable fragment”), providing they have retained the original bindingproperties, form part of the present invention. Such fragments arecommonly generated by, for instance, enzymatic digestion of theantibodies with papain, pepsin, or other proteases. It is well known tothe person skilled in the art that monoclonal antibodies, or fragmentsthereof, can be modified for various uses. The antibodies involved inthe invention can be labeled by an appropriate label of the enzymatic,fluorescent, or radioactive type.

Small molecules, e.g., small organic molecules, and other drugcandidates can be obtained, for example, from combinatorial and naturalproduct libraries. To screen for the candidate/test molecules, celllines that express VEGFR-1 and VEGFR-2 may be used and the signaltransduction is monitored as described in detail in the examples.

Monitoring can be measured using standard biochemical techniques. Otherresponses, such as activation or suppression of catalytic activity,phosphorylation (e.g., the tyrosine phosphorylation of the intracellulardomain of VEGFR-2) or dephosphorylation of other proteins, activation ormodulation of second messenger production, changes in cellular ionlevels, association, dissociation or translocation of signalingmolecules, or transcription or translation of specific genes, may alsobe monitored. These assays may be performed using conventionaltechniques developed for these purposes in the course of screening.Inhibition of ligand binding to its cellular receptor may, via signaltransduction pathways, affect a variety of cellular processes.

Cellular processes under the control of the VEGFR-1/PIGF signalingpathway may include, but are not limited to, normal cellular functions,proliferation, differentiation, maintenance of cell shape, and adhesion,in addition to abnormal or potentially deleterious processes such asunregulated cell proliferation, loss of contact inhibition, blocking ofdifferentiation or cell death. The qualitative or quantitativeobservation and measurement of any of the described cellular processesby techniques known in the art may be advantageously used as a means ofscoring for signal transduction in the course of screening.

Random peptide libraries, such as tetrameric peptide libraries furtherdescribed herein, consisting of all possible combinations of amino acidsattached to a solid phase support, may be used to identify peptides thatare able to bind to the ligand binding site of a given receptor or otherfunctional domains of a receptor such as kinase domains (K. S. Lam etal., 1991, Nature 354, 82). The screening of peptide libraries may havetherapeutic value in the discovery of pharmaceutical agents that act toinhibit the biological activity of receptors through their interactionswith the given receptor.

Identification of molecules that are able to bind to the VEGFR-1 or PIGFmay be accomplished by screening a peptide library with recombinantsoluble VEGFR-1 protein or PIGF protein. For example, the kinase andextracellular ligand binding domains of VEGFR-1 may be separatelyexpressed and used to screen peptide libraries. In addition to usingsoluble VEGFR-1 molecules, in another embodiment, it is possible todetect peptides that bind to cell surface receptors using intact cells.The cells used in this technique may be either alive or fixed cells. Thecells will be incubated with the random peptide library and will bindcertain peptides in the library to form a “rosette” between the targetcells and the relevant solid phase support/peptide. The rosette canthereafter be isolated by differential centrifugation or removedphysically under a dissecting microscope.

In another embodiment, transdominant-negative mutant forms of VEGFreceptors (e.g., a transdominant-negative receptor of VEGFR-1) can beused to inhibit the signal transduction of PIGF. The use of thetransdominant-negative mutant forms of VEGF receptors is fully describedin U.S. Pat. No. 5,851,999.

Also within the scope of the invention are oligoribonucleotide sequencesthat include antisense RNA and DNA molecules and ribozymes that functionto inhibit the translation of VEGFR-1 mRNA or PIGF mRNA. Antisense RNAand DNA molecules act to directly block the translation of mRNA bybinding to targeted mRNA and preventing protein translation. In regardto antisense DNA, oligodeoxyribonucleotides derived from the translationinitiation site, e.g., between −10 and +10 regions of the VEGFR-1 orPIGF nucleotide sequence, are preferred. Ribozymes are enzymatic RNAmolecules capable of catalyzing the specific cleavage of RNA. Themechanism of ribozyme action involves sequence-specific hybridization ofthe ribozyme molecule to complementary target RNA, followed by anendonucleolytic cleavage. Within the scope of the invention areengineered hammerhead motif ribozyme molecules that specifically andefficiently catalyze endonucleolytic cleavage of VEGFR-1 or PIGF RNAsequences.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites that include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for predicted structuralfeatures such as secondary structures that may render theoligonucleotide sequence unsuitable. The suitability of candidatetargets may also be evaluated by testing their accessibility tohybridization with complementary oligonucleotides, using ribonucleaseprotection assays.

Both antisense RNA and DNA molecules and ribozymes of the invention maybe prepared by any method known in the art for the synthesis of RNAmolecules. These include techniques for chemically synthesizingoligodeoxyribonucleotides well known in the art, such as, for example,solid phase phosphoramidite chemical synthesis.

Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding the antisense RNA molecule. SuchDNA sequences may be incorporated into a wide variety of vectors thatincorporate suitable RNA polymerase promoters such as the T7 or SP6polymerase promoters. Alternatively, antisense cDNA constructs thatsynthesize antisense RNA constitutively or inducibly, depending on thepromoter used, can be stably introduced into cell lines.

In another embodiment of the invention, the above-described moleculescan be used as a medicament to treat pathological conditions ofangiogenesis and/or arteriogenesis and/or edema formation.

“Edema” is a condition that is caused by vascular leakage. Vasodilationand increased permeability during inflammation can be predominantpathogenetic mechanisms. For instance, edema contributes to infarctexpansion after stroke and may cause life-threatening intracranialhypertension in cancer patients. Further, extravasation of plasmaproteins favors metastatic spread of occult tumors, and airwaycongestion may cause fatal asthmatic attacks. The increased vascularleakage that occurs during inflammation can lead to respiratorydistress, ascites, peritoneal sclerosis (in dialysis patients), adhesionformation (abdominal surgery) and metastatic spreading.

By “angiogenesis,” it is meant a fundamental process by which new bloodvessels are formed. The primary angiogenic period in humans takes placeduring the first three months of embryonic development, but angiogenesisalso occurs as a normal physiological process during periods of tissuegrowth, such as an increase in muscle or fat and during the menstrualcycle and pregnancy. The term “pathological angiogenesis” refers to theformation and growth of blood vessels during the maintenance and theprogression of several disease states, for example, in blood vessels(atherosclerosis, hemangioma, hemangioendothelioma), bone and joints(rheumatoid arthritis, synovitis, bone and cartilage destruction,osteomyelitis, pannus growth, osteophyte formation, neoplasms andmetastasis), skin (warts, pyogenic granulomas, hair growth, Kaposi'ssarcoma, scar keloids, allergic edema, neoplasms), liver, kidney, lung,ear and other epithelia (inflammatory and infectious processes(including hepatitis, glomerulonephritis, pneumonia), asthma, nasalpolyps, otitis, transplantation, liver regeneration, neoplasms andmetastasis), uterus, ovary and placenta (dysfunctional uterine bleeding(due to intra-uterine contraceptive devices), follicular cyst formation,ovarian hyperstimulation syndrome, endometriosis, neoplasms), brain,nerves and eye (retinopathy of prematurity, diabetic retinopathy,choroidal and other intraocular disorders, leukomalacia, neoplasms andmetastasis), heart and skeletal muscle due to work overload, adiposetissue (obesity), endocrine organs (thyroiditis, thyroid enlargement,pancreas transplantation), hematopoiesis (AIDS (Kaposi), hematologicmalignancies (leukemias, etc.), lymph vessels (tumor metastasis,lymphoproliferative disorders).

By “retinal ischemic diseases,” it is meant that the retina's supply ofblood and oxygen is decreased, and the peripheral portions of the retinalose their source of nutrition and stop functioning properly. Commondiseases that lead to retinopathy are diabetic retinopathy, centralretinal vein occlusion, stenosis of the carotid artery, and sickle cellretinopathy. Diabetic retinopathy is a major cause of visual loss indiabetic patients. In the ischemic retina, the growth of new bloodvessels occurs (neovascularization). These vessels often grow on thesurface of the retina, at the optic nerve, or in the front of the eye onthe iris. The new vessels cannot replace the flow of necessary nutrientsand, instead, can cause many problems such as vitreous hemorrhage,retinal detachment, and uncontrolled glaucoma. These problems occurbecause new vessels are fragile and are prone to bleed. If caught in itsearly stages, proliferative diabetic retinopathy can sometimes bearrested with panretinal photocoagulation. However, in some cases,vitrectomy surgery is the only option.

By the term “pulmonary hypertension,” it is meant a disorder in whichthe blood pressure in the pulmonary arteries is abnormally high. In theabsence of other diseases of the heart or lungs, it is called primarypulmonary hypertension. Diffuse narrowing of the pulmonary arteriolesoccurs as a result of pathological arteriogenesis followed by pulmonaryhypertension as a response to the increased resistance to blood flow.The incidence is eight out of 100,000 people. However, pulmonaryhypertension can also occur as a complication of Chronic ObstructivePulmonary Diseases (COPD) such as emphysema, chronic bronchitis ordiffuse interstitial fibrosis and in patients with asthmatiform COPD.The incidence of COPD is approximately five out of 10,000 people.

In another embodiment of the invention, the above-described moleculescan be used to manufacture a medicament to treat inflammation and, morespecifically, inflammatory disorders. “Inflammation,” as used herein,means the local reaction to injury of living tissues, especially thelocal reaction of the small blood vessels, their contents, and theirassociated structures. The passage of blood constituents through thevessel walls into the tissues is the hallmark of inflammation, and thetissue collection so formed is termed the “exudates” or “edema.” Anynoxious process that damages living tissue (infection with bacteria,excessive heat, cold, mechanical injury such as crushing, acids,alkalis, irradiation, or infection with viruses) can cause inflammationirrespective of the organ or tissue involved. It should be clear thatdiseases of animals and man classed as “inflammatory diseases” andtissue reactions ranging from burns to pneumonia, leprosy, tuberculosis,and rheumatoid arthritis are all “inflammations.”

In another embodiment of the invention, the above-described moleculescan be used to manufacture a medicament to treat tumor formation. By“tumor,” it is meant a mass of abnormal tissue that arises withoutobvious cause from pre-existing body cells, has no purposeful function,and is characterized by a tendency to autonomous and unrestrainedgrowth. Tumors are quite different from inflammatory or other swellingsbecause the cells in tumors are abnormal in their appearance and othercharacteristics. Abnormal cells, the kind that generally make up tumors,differ from normal cells in that they have undergone one or more of thefollowing alterations: (1) hypertrophy, or an increase in the size ofindividual cells; this feature is occasionally encountered in tumors butoccurs commonly in other conditions; (2) hyperplasia or an increase inthe number of cells within a given zone; in some instances, it mayconstitute the only criterion of tumor formation; (3) anaplasia, or aregression of the physical characteristics of a cell toward a moreprimitive or undifferentiated type; this is an almost constant featureof malignant tumors, though it occurs in other instances both in healthand in disease. In some instances, the cells of a tumor are normal inappearance, faithful reproductions of their parent types; thedifferences between them and normal body cells are difficult to discern.Such tumors are also often benign.

Other tumors are composed of cells that appear different from normaladult types in size, shape, and structure; they usually belong to tumorsthat are malignant. Such cells may be bizarre in form or be arranged ina distorted manner. In more extreme cases, the cells of malignant tumorsare described as primitive, or undifferentiated, because they have lostthe appearance and functions of the particular type of (normal)specialized cell that was their predecessor. As a rule, the lessdifferentiated a malignant tumor's cells are, the more quickly thattumor may grow. Malignancy refers to the ability of a tumor toultimately cause death. Any tumor, either benign or malignant in type,may produce death by local effects if it is “appropriately” situated.

The common and more specific definition of malignancy implies aninherent tendency of the tumor's cells to metastasize (invade the bodywidely and become disseminated by subtle means) and eventually to killthe patient unless all the malignant cells can be eradicated. Metastasisis thus the outstanding characteristic of malignancy. Metastasis is thetendency of tumor cells to be carried from their site of origin by wayof the circulatory system and other channels, which may eventuallyestablish these cells in almost every tissue and organ of the body. Incontrast, the cells of a benign tumor invariably remain in contact witheach other in one solid mass centered on the site of origin. Because ofthe physical continuity of benign tumor cells, they may be removedcompletely by surgery if the location is suitable. But the disseminationof malignant cells, each one individually possessing (through celldivision) the ability to give rise to new masses of cells (new tumors)in new and distant sites, precludes complete eradication by a singlesurgical procedure in all but the earliest period of growth. A benigntumor may undergo malignant transformation, but the cause of such changeis unknown. It is also possible for a malignant tumor to remainquiescent, mimicking a benign one clinically, for a long time. Allbenign tumors tend to remain localized at the site of origin. Manybenign tumors are encapsulated. The capsule consists of connectivetissue derived from the structures immediately surrounding the tumor.

Well-encapsulated tumors are not anchored to their surrounding tissues.These benign tumors enlarge by accretion, pushing aside the adjacenttissues without involving them intimately. Among the major types ofbenign tumors are the following: lipomas, which are composed of fatcells; angiomas, which are composed of blood or lymphatic vessels;osteomas, which arise from bone; chondromas, which arise from cartilage;and adenomas, which arise from glands. For malignant tumors, examplescomprise carcinomas (occur in epithelial tissues, which cover the body(the skin) and line the inner cavitary structures of organs (such as thebreast, the respiratory and gastrointestinal tracts), the endocrineglands, and the genitourinary system) and sarcomas that develop inconnective tissues, including fibrous tissues, adipose (fat) tissues,muscle, blood vessels, bone, and cartilage. A cancer can also develop inboth epithelial and connective tissue and is called a carcinosarcoma.Cancers of the blood-forming tissues (such as leukemias and lymphomas),tumors of nerve tissues (including the brain), and melanoma (a cancer ofthe pigmented skin cells) are classified separately.

In a specific embodiment, it should be clear that the therapeutic methodof the present invention against tumors can also be used in combinationwith any other tumor therapy known in the art such as irradiation,chemotherapy or surgery.

The term “medicament to treat” relates to a composition comprisingmolecules as described above and a pharmaceutically acceptable carrieror excipient (both terms can be used interchangeably) to treat diseasesas indicated above. Suitable carriers or excipients known to the skilledman are saline, Ringer's solution, dextrose solution, Hank's solution,fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhanceisotonicity and chemical stability, buffers and preservatives. Othersuitable carriers include any carrier that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids and amino acid copolymers.

The “medicament” may be administered by any suitable method within theknowledge of the skilled man. The preferred route of administration isparenterally. In parental administration, the medicament of thisinvention will be formulated in a unit dosage injectable form such as asolution, suspension or emulsion, in association with thepharmaceutically acceptable excipients as defined above. However, thedosage and mode of administration will depend on the individual.Generally, the medicament is administered so that the protein,polypeptide, or peptide of the present invention is given at a dosebetween 1 μg/kg and 10 mg/kg, more preferably between 10 μg/kg and 5mg/kg, most preferably between 0.1 and 2 mg/kg. Preferably, it is givenas a bolus dose. Continuous infusion may also be used and includescontinuous subcutaneous delivery via an osmotic minipump. If so, themedicament may be infused at a dose between 5 and 20 μg/kg/minute, morepreferably between 7 and 15 μg/kg/minute.

In another embodiment, antibodies or functional fragments thereof can beused for the manufacture of a medicament for the treatment of theabove-mentioned disorders. Non-limiting examples are the commerciallyavailable goat polyclonal antibody from R&D Pharmaceuticals, Abingdon,UK, or the chicken polyclonal antibody (Gassmann et al., 1990, Faseb J.4, 2528). Preferentially, the antibodies are humanized (Rader et al.,2000, J. Biol. Chem. 275, 13668) and, more preferentially, humanantibodies are used as a medicament.

Another aspect of administration for treatment is the use of genetherapy to deliver the above-mentioned antisense gene or functionalparts of the PIGF gene or a ribozyme directed against the PIGF mRNA or afunctional part thereof. “Gene therapy” means the treatment by thedelivery of therapeutic nucleic acids to a patient's cells. This isextensively reviewed in Lever and Goodfellow 1995; Br. Med. Bull. 51,1-242; Culver 1995; F. D. Ledley, 1995, Hum. Gene Ther. 6, 1129. Toachieve gene therapy, there must be a method of delivering genes to thepatient's cells and additional methods to ensure the effectiveproduction of any therapeutic genes. There are two general approaches toachieve gene delivery; these are non-viral delivery and virus-mediatedgene delivery.

In another embodiment of the invention, a molecule to inhibit theactivity of PIGF, as described above, can be used in combination with amolecule to inhibit the activity of VEGF, according to the sameinhibition levels as described above for PIGF. Indeed, PIGF is found tobe angiogenic at sites where VEGF levels are increased.

In another embodiment, the invention provides a method to identifymolecules that can interfere with the binding of PIGF to the VEGFReceptor-1 (VEGFR-1). This method comprises exposing PIGF or VEGFR-1 toat least one molecule and measuring the ability of at least one moleculeto interfere with the binding of PIGF to VEGFR-1 and monitoring theability of at least one molecule to prevent or to inhibit pathologicalangiogenesis, vascular leakage, pulmonary hypertension, tumor formationand/or inflammatory disorders.

In another embodiment, the invention provides a method to identifymolecules comprising: exposing placental growth factor or vascularendothelial growth factor receptor-1 and/or neuropilin-1 or nucleicacids encoding the growth factor to at least one molecule whose abilityto suppress or prevent placental growth factor-induced pathologicalangiogenesis, vascular leakage (edema), pulmonary hypertension, tumorformation and/or inflammatory disorders is sought to be determined, andmonitoring the pathological angiogenesis, vascular leakage (edema),pulmonary hypertension, tumor formation and/or inflammatory disorders.

The invention also provides methods for identifying compounds ormolecules that bind on the VEGFR-1 or on PIGF and prevent theinteraction between PIGF and VEGFR-1 and consequently are able toantagonize the signal transduction. The latter methods are also referredto as “drug screening assays” or “bioassays” and typically include thestep of screening a candidate/test compound or agent for the ability tointeract with VEGFR-1 or PIGF. Candidate compounds or agents that havethis ability can be used as drugs to combat or prevent pathologicalconditions of angiogenesis, arteriogenesis or edema formation.Candidate/test compounds, such as small molecules, e.g., small organicmolecules, and other drug candidates can be obtained, for example, fromcombinatorial and natural product libraries as described above.

Typically, the assays are cell-free assays, which include the steps ofcombining VEGFR-1 or PIGF and a candidate/test compound, e.g., underconditions that allow for interaction of (e.g., binding of) thecandidate/test compound with VEGFR-1 or PIGF to form a complex, anddetecting the formation of a complex in which the ability of thecandidate compound to interact with VEGFR-1 or PIGF is indicated by thepresence of the candidate compound in the complex. Formation ofcomplexes between the VEGFR-1 or PIGF and the candidate compound can bequantitated, for example, using standard immunoassays. The VEGFR-1 orPIGF employed in such a test may be free in solution, affixed to a solidsupport, born on a cell surface, or located intracellularly.

To perform the above-described drug screening assays, it is feasible toimmobilize VEGFR-1 or PIGF or its (their) target molecule(s) tofacilitate separation of complexes from uncomplexed forms of one or bothof the proteins, as well as to accommodate automation of the assay.Interaction (e.g., binding of) of VEGFR-1 or PIGF to a target moleculecan be accomplished in any vessel suitable for containing the reactants.

Examples of such vessels include microtiter plates, test tubes, andmicrocentrifuge tubes. In one embodiment, a fusion protein can beprovided that adds a domain that allows the protein to be bound to amatrix. For example, VEGFR-1-His or PIGF tagged can be adsorbed ontoNi-NTA microtiter plates, or VEGFR-1-ProtA or PIGF fusions adsorbed toIgG, which are then combined with the cell lysates (e.g., ³⁵S-labeled)and the candidate compound, and the mixture incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the plates are washed to remove anyunbound label, and the matrix immobilized and radiolabel determineddirectly, or in the supernatant after the complexes are dissociated.Alternatively, the complexes can be dissociated from the matrix andseparated by SDS-PAGE, and the level of VEGFR-1 or PIGF binding proteinfound in the bead fraction quantitated from the gel using standardelectrophoretic techniques. Other techniques for immobilizing protein onmatrices can also be used in the drug screening assays of the invention.For example, either VEGFR-1 or PIGF and VEGFR-1 or its target moleculescan be immobilized utilizing conjugation of biotin and streptavidin.Biotinylated VEGFR-1 or PIGF can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.) and immobilized inthe wells of streptavidin-coated 96-well plates (Pierce Chemical).Alternatively, antibodies reactive with VEGFR-1 or PIGF, but which donot interfere with binding of the protein to its target molecule, can bederivatized to the wells of the plate, and VEGFR-1 or PIGF trapped inthe wells by antibody conjugation. As described above, preparations of aVEGFR-1-binding protein or PIGF and a candidate compound are incubatedin the VEGFR-1 or PIGF-presenting wells of the plate, and the amount ofcomplex trapped in each well can be quantitated.

Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the VEGFR-1-target molecule orPIGF, or reactive with VEGFR-1 or PIGF and compete with the targetmolecule, as well as enzyme-linked assays that rely on detecting anenzymatic activity associated with the target molecule. Anothertechnique for drug screening that provides for high throughput screeningof compounds having suitable binding affinity to VEGFR-1 or PIGF isdescribed in detail in “Determination of Amino Acid SequenceAntigenicity” by H. N. Geysen, WO 84/03564, published on Sep. 13, 1984.In summary, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. The protein test compounds are reacted with fragments ofVEGFR-1 or PIGF and washed. Bound VEGFR-1 or PIGF is then detected bymethods well known in the art. Purified VEGFR-1 or PIGF can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support. This inventionalso contemplates the use of competitive drug screening assays in whichneutralizing antibodies capable of binding VEGFR-1 or PIGF specificallycompete with a test compound for binding VEGFR-1 or PIGF. In thismanner, the antibodies can be used to detect the presence of anyprotein, which shares one or more antigenic determinants with VEGFR-1 orPIGF.

In another embodiment, the invention provides a method for theproduction of a pharmaceutical composition comprising the usage of themethod according to claims 6-7 and further mixing the moleculeidentified, or a derivative or homologue thereof, with apharmaceutically acceptable carrier.

In another embodiment, PIGF promoter polymorphisms can be used toidentify individuals having a predisposition to acquire pathologicalangiogenesis, vascular leakage (edema), pulmonary hypertension, tumorformation and/or inflammatory disorders. Indeed, it can be expected thatpromoter polymorphisms can give rise to much higher or much lower levelsof PIGF. Consequently, higher levels of PIGF can lead to apredisposition to acquire pathological angiogenesis, vascular leakage(edema), pulmonary hypertension, tumor formation and/or inflammatorydisorders while much lower levels of PIGF can lead to a protection toacquire pathological angiogenesis, vascular leakage (edema), pulmonaryhypertension, tumor formation and/or inflammatory disorders.

The following examples more fully illustrate preferred features of theinvention, but are not intended to limit the invention in any way. Allof the starting materials and reagents disclosed below are known tothose skilled in the art, and are available commercially or can beprepared using well-known techniques.

EXAMPLES 1. Impaired Pathological Angiogenesis in PIGF^(−/−) Mice

In several pathological conditions, in particular when associated withincreased VEGF expression, formation of new endothelial-lined channels(angiogenesis) was significantly impaired in PIGF^(−/−) mice. Growth andangiogenesis of embryonic stem (ES) cell-derived tumors, known to bemediated by VEGF (N. Ferrara et al., 1996, Nature 380, 439-442), wasalso dependent on PIGF. Indeed, PIGF^(+/+) ES cell-derived tumorsobtained within four weeks after subcutaneous inoculation in nu/nuPIGF^(+/+) mice, weighed 4±1 g (n=8) and appeared hemorrhagic and bledprofusely (seven of eight tumors). In contrast, PIGF^(−/−) tumors innu/nu PIGF^(−/−) hosts only weighed 1.0±0.3 g (n=8) and werehomogeneously white with minimal bleeding (five of seven tumors). Growthand vascularization in PIGF^(−/−) tumors were reduced to the same degreeas in VEGF^(−/−) tumors. PIGF^(+/+) and PIGF^(−/−) tumors containedcomparable vascular densities of endothelial cords and capillaries(diameter <8 μm). However, compared to PIGF^(+/+) tumors PIGF^(−/−)tumors contained fewer medium-sized or large vessels. In a previousstudy, we demonstrated that formation of medium-sized and large vesselsis dependent on VEGF (P. Carmeliet et al., 1998, Nature 394, 485-490).Angiogenesis of PIGF^(+/+) tumors in nu/nu PIGF^(−/−) mice or ofPIGF^(−/−) tumors in nu/nu PIGF^(+/+) mice was comparable to that ofPIGF^(+/+) tumors in nu/nu PIGF^(+/+) mice, indicating that productionof PIGF, either by tumor or by host-derived tissue, could rescue thephenotype. VEGF transcript levels were comparable between both genotypes(VEGF/10³ HPRT mRNA molecules: 280±20 in PIGF^(+/+) tumors versus 320±50in PIGF^(−/−) tumors; p=NS) and were expressed in epithelial andmesenchymal cells throughout PIGF^(+/+) tumors, whereas PIGF wasexpressed in endothelial cells of small and large vessels (in situhybridization). Expression of VEGF-B and VEGF-C was also comparable.

Exposure of neonatal mice to 80% oxygen from P7 to P12 causes capillarydropout in the retina due to reduced VEGF expression (VEGF/10³ HPRT mRNAmolecules in PIGF^(+/+) retinas: 270±40 at P7 during normoxia versus100±10 at P12 during hyperoxia; p<0.05 versus P7; n=5) (T. Alon et al.,1995, Nat. Med. 1, 1024-1028). Upon reexposure to room air at P12,retinal ischemia up-regulates VEGF expression (VEGF/10³ HPRT mRNAmolecules: 390±50 at P13, p<0.05 versus P7; and 165±50 at P17; n=5),thereby inducing venous dilatation, arterial tortuosity, and capillaryoutgrowth in the vitreous chamber by P17 (T. S. Kern et al., 1996, Arch.Opthalmol. 114, 986-990). The role of PIGF in ischemic retinopathyremains unknown. PLGF transcript levels (PIGF/10³ HPRT mRNA molecules)were 40±10 at P7 (normoxia), 10±2 at P12 (hyperoxia), 90±10 at P13(return to normoxia) and 12±2 at P17. Despite a comparable retinalvascular development during normoxia and a comparable capillary dropoutduring hyperoxia (P12) in both genotypes, PIGF^(−/−) mice developed ˜75%fewer and significantly smaller neovascular vitreous tufts by P17 thanPIGF^(+/+) mice (endothelial cells per section: 10±2 in PIGF^(−/−) miceversus 48±4 in PIGF^(+/+) mice; n=6; p<0.05). In addition, PIGF^(−/−)mice exhibited reduced venous dilatation (semiquantitative dilatationscore, see methods: 0.9±0.05 in PIGF^(−/−) mice versus 1.7±0.03 inPIGF^(+/+) mice; p<0.05) and arterial tortuosity (tortuosity score:0.8±0.05 in PIGF^(−/−) mice versus 2.3±0.2 in PIGF^(+/+) mice; p<0.05).

2. Reduced Vascular Permeability in PIGF^(−/−) Mice

Vascular permeability, a characteristic feature of VEGF (N. Ferrara etal., 1999, Curr. Top. Microbiol. Immunol. 237, 1-30), was consistentlyreduced in PIGF^(−/−) mice as compared to PIGF^(+/+) mice. VEGF has beenpreviously implicated in vascular permeability of the skin (L. F. Brownet al., 1995, J. Immunol. 154, 2801-2807), but the role of PIGF remainsundetermined. Several models were used: (i) Intradermal injection of 1,3 or 10 ng VEGF₁₆₅ induced less extravasation of Evans blue inPIGF^(−/−) mice than in PIGF^(+/+) mice (Miles assay). (ii) Skinsensitization with ovalbumin caused less extravasation of plasma inPIGF^(−/−) mice than in PIGF^(+/+) mice (J. Casals-Stenzel et al., 1987,Immunopharmacology 13, 177-183) (Arthus reaction: 12±1 μl extravasatedplasma after vehicle versus 130±5 μl plasma after ovalbumin in PIG^(+/+)mice; p<0.05; n=12 as compared to 11±1 μl plasma after vehicle versus13±1 μl plasma after ovalbumin in PIGF^(−/−) mice; p=NS; n=12). (iii)Plasma extravasation in normal skin vessels was similar in bothgenotypes (mg plasma×10⁵/min. mg tissue: 35±5 in PIGF^(+/+) mice versus35±4 in PIGF^(−/−) mice; p=NS; n=7) but increased significantly more inPIGF^(+/+) than in PIGF^(−/−) mice after skin wounding (60±4 inPIGF^(+/+) mice versus 40±4 in PIGF^(−/−) mice; p<0.05; n=10). Thus,PIGF specifically increased the vascular permeability in response toVEGF, but not to histamine.

3. Impaired Pathological Arteriogenesis in PIGF^(−/−) Mice

Healing of skin wounds is mediated by ingrowth of vessels, whichinitially consist of endothelial cells (angiogenesis) and subsequentlybecome surrounded by smooth muscle cells (arteriogenesis). VEGF has beenimplicated in capillary growth during skin healing (M. Detmar et al.,1998, J. Invest. Dermatol. 111, 1-6), but the role of PIGF remainsunknown. Healing of skin incisions was slightly retarded in PIGF^(−/−)as compared to PIGF^(+/+) mice. Both genotypes contained comparabledensities of thrombomodulin-stained vessels in unwounded skin(vessels/mm²: 240±80 in PIGF^(+/+) mice versus 200±80 in PIGF^(−/−)mice; p=NS; n=5). Smooth muscle α-actin staining revealed a comparabledensity of vessels (i) that were not covered or surrounded by a fewsmooth muscle cells (vessels/mm²: 58±14 in PLGF^(+/+) mice versus 40±12in PIGF^(−/−) mice; p=NS; n=5), and (ii) that were completely covered byat least one smooth muscle cell layer (vessels/mm²: 15±5 in PIGF^(+/+)mice versus 19±1 in PIGF^(−/−) mice; p=NS; n=5). Within four days afterwounding, PIGF was expressed in endothelial cells, and PIGF and VEGFwere up-regulated in PIGF^(+/+) keratinocytes in the hyperplasticepidermis at the wound edge, where new vessels formed (in situhybridization; not shown). Both strains contained comparable vesselingrowth in the wound region (thrombomodulin-stained vessels/mm²: 240±50in PIGF^(+/+) mice versus 180±50 in PIGF^(−/−) mice; n=5; p=NS).However, both genotypes differed in the degree the new vessels werecovered by SMA-positive smooth muscle cells. The number of vessels thatwere not or incompletely covered by smooth muscle cells was 40±7 inPIGF^(+/+) mice versus 84±13 in PIGF^(−/−) mice (p<0.05; n=5), whereasthe number of vessels that were completely covered by at least onesmooth muscle cell layer was 75±18 in PIGF^(+/+) mice versus 30±10 inPIGF^(−/−) mice (p<0.05). Thus, lack of PIGF impairs coverage of newendothelial channels with smooth muscle cells.

Pulmonary hypertension due to hypoxia-induced remodeling of thepulmonary vasculature is a life-threatening complication of chronicobstructive pulmonary disease (COPD). Even though VEGF is highlyup-regulated in lungs of patients with COPD (C. D. Cool et al., 1999,Am. J. Pathol. 155, 411-419) and of hypoxic animals (H. Christou et al.,1998, Am. J. Respir. Cell. Mol. Biol. 18, 768-776), its role in thisprocess is not understood. Surprisingly, no information is availableabout the expression or role of PIGF. Therefore, adult mice were exposedto hypoxia (10% O₂) during four weeks, as this causes pulmonaryhypertension due to increased “muscularization” of the pulmonary vessels(C. A. Hales et al., 1983, Am. Rev. Respir. Dis. 128, 747-751). Theratio of the right ventricular (R V) to left ventricular (LV) weight (ameasure of RV hypertrophy) was comparable in both genotypes duringnornoxia (32±2% in PIGF^(+/+) mice versus 33±2% in PIGF^(−/−) mice; n=4;p=NS), significantly increased after hypoxia in PIGF^(+/+) mice (48±4%;n=5; p<0.05 versus normoxia), but only minimally affected by hypoxia inPIGF^(−/−) mice (37±2%; n=6; p<0.05 versus normoxia and versusPIGF^(+/+)). Significant genotypic differences in pulmonary vascularremodeling were observed. Elastin staining of normoxic lungs revealedthat both genotypes had a comparable density of intra-acinar thin-walledarterioles containing only an internal elastic lamina (IEL), orthick-walled arterioles containing an intact IEL plus an incompleteexternal elastic lamina (EEL) (Table 1). Thick-walled arteriolescontaining two intact elastic laminae over their entire circumferencewere only occasionally detected in both genotypes (Table 1). Hypoxiacaused significant vascular remodeling in PIGF^(+/+) mice, resulting ina larger fraction of thick-walled vessels with a partial or complete EELat the expense of thin-walled vessels with only a single IEL (Table 1).In contrast, vascular remodeling was much less significant in PIGF^(−/−)mice, resulting in a smaller fraction of thick-walled vessels with acomplete IEL and EEL (Table 1). Immunostaining for smooth muscle α-actin(SMA) confirmed that PIGF^(+/+) mice contained significantly more fullymuscularized arterioles than PIGF^(−/−) mice after hypoxia (Table 1).Protection against pulmonary hypertension in PIGF^(−/−) mice was not dueto a reduced vasoconstriction response (RV pressure increased by 31±4%in PIGF^(+/+) mice versus 34±5% in PIGF^(−/−) mice in response to 30minutes 7% O₂; p=NS), nor was it due to lower hematocrit levels (48±3%in PIGF^(+/+) mice versus 53±3% in PIGF^(−/−) mice; p=NS). Thus, PIGFsignificantly modulates arterial remodeling.

TABLE 1 Pulmonary vascular remodeling after chronic hypoxia. Vessels per10³ alveoli PLGF^(+/+) mice PLGF^(−/−) mice 20% O₂ 10% O₂ 20% O₂ 10% O₂Presence of elastic laminae Single IEL 11 ± 2 3.6 ± 0.6* 11 ± 1 5.6 ±0.6*, # IEL + incomplete 11 ± 2  12 ± 1 10 ± 2  11 ± 1 EEL IEL +complete <0.5   6 ± 1* <0.5   3 ± 1*, # EEL Coverage by SMC Absent  2.4± 0.9 0.5 ± 0.3*  3.1 ± 0.7 3.4 ± 0.7* Incomplete 11 ± 1  12 ± 2  8 ± 2 11 ± 1 Complete  1.2 ± 0.5  11 ± 2*, # 2.6 ± 2  2.8 ± 2*, #

The data represent the number (average ±SEM in five mice) of vessels per10³ alveoli containing a single internal elastic lamina (EEL), an IELplus an incomplete external elastic lamina (EEL), or an IEL plus acomplete EEL. In addition, the density of vessels that were not(absent), incompletely or completely surrounded by smooth muscle α-actinstained smooth muscle cells (SMC) is shown. *: p<0.05 versus normoxia(20% O₂); #: p<0.05 versus PLGF^(+/+).

4. Synergism Between PIGF and VEGF

Proliferation and survival of endothelial cells in response to VEGF werestudied. VEGF₁₆₅ stimulated proliferation of PIGF^(+/+) endothelialcells (Table 2) and protected them against apoptosis induced by serumdeprivation (0.1% serum) or supplementation of TNF-α (10 ng/ml). Incontrast, VEGF₁₆₅ failed to stimulate proliferation or to protectPIGF^(−/−) endothelial cells against serum deprivation- or TNF-α-inducedapoptosis (Table 2). PIGF itself was not mitogenic nor anti-apoptoticfor endothelial cells of either genotype. Also, it did not affect theresponse of PIGF^(+/+) endothelial cells to VEGF (Table 2), likelybecause PIGF^(+/+) endothelial cells already produced sufficient PIGF(the variable degree of PLGF production by endothelial cells and therelative amounts of VEGF present in the culture conditions may in factexplain why some, but not others, have previously observed an angiogenicresponse in vitro). However, PIGF rescued the impaired proliferation andsurvival response of PIGF^(−/−) endothelial cells to VEGF₁₆₅ (Table 2).PIGF was also found to modulate the mitogenic response to VEGF of smoothmuscle cells, known to express VEGFR-1 and VEGFR-2 (C. L. Grosskreutz etal., 1999, Microvasc. Res. 58, 128-136). Indeed, VEGF stimulatedproliferation of PIGF^(+/+) but not of PIGF^(−/−) smooth muscle cells.PIGF, ineffective by itself, restored the responsiveness of PIGF^(−/−)cells to VEGF. PIGF specifically modulated the activity of VEGF, sincePIGF^(−/−) and PIGF^(+/+) cells displayed comparable responses to bFGF.Thus, PIGF affected endothelial and smooth muscle cells only when theywere stimulated with VEGF.

The mechanism found to play a role in the synergism between VEGF andPIGF: (i) PIGF up-regulated the expression of VEGF, as previouslysuggested (M. J. Bottomley et al., 2000, Clin. Exp. Immunol. 119,182-188). Expression of VEGF by PIGF^(−/−) fibroblasts was increased byPIGF (VEGF production per 10⁶ cells/ml/24 hours: 180±10 pg aftertreatment with vehicle versus 440±10 pg after treatment with 100 ng/mlPIGF for 48 hours; p<0.05 versus vehicle). Similar results were obtainedfor PIGF^(+/+) fibroblasts (VEGF production per 10⁶ cells/ml/24 hours:200±8 pg after treatment with vehicle versus 430±5 pg after treatmentwith 100 ng/ml PIGF for 48 hours; p<0.05 versus vehicle).

Induction of VEGF production by PIGF was, however, smaller than thatinduced by hypoxia (1% O₂) (expressed per 10⁶ cells/ml/24 hours:3200±150 pg for PIGF^(+/+) cells; 2400±150 pg for PIGF^(−/−) cells;p<0.05 versus normoxia). VEGF immunoreactivity was also increased inPIGF^(+/+) mice after treatment with 1.5 μg PIGF₁₃₂/24 hours.

TABLE 2 Role of PLGF and VEGF in endothelial proliferation. Endothelialproliferation PLGF^(+/+) PLGF^(−/−) Vehicle 11 ± 2  11 ± 1 VEGF¹²⁰ (100ng/ml) 22 ± 1* 12 ± 1 VEGF¹⁶⁵ (100 ng/ml) 36 ± 4* 12 ± 2 VEGF¹⁶⁵ (300ng/ml) 42 ± 3* 13 ± 3 VEGF-E (100 ng/ml) 32 ± 1* 13 ± 1 PLGF (100 ng/ml)11 ± 1  11 ± 1 VEGF¹⁶⁵ (100 ng/ml) + PLGF (100 ng/ml) 33 ± 3*  33 ± 2*VEGF¹⁶⁵ (100 ng/ml) +anti-NP1 MoAb 27 ± 3* N.D. +anti-NP2 MoAb 34 ± 3 N.D. +anti-VEGFR-1 Ab 23 ± 5* 12 ± 2 +anti-VEGFR-2 MoAb 15 ± 2  N.D.VEGF¹⁶⁵ (100 ng/ml) + PLGF (100 ng/ml) +anti-VEGFR-1 Ab 22 ± 4  21 ± 4+anti-VEGFR-2 MoAb 13 ± 3  17 ± 2 bFGF (50 ng/ml) 35 ± 2*  35 ± 5* bFGF(50 ng/ml) + PLGF (100 ng/ml) 32 ± 3*  33 ± 1*

The data represent the mean±SD of nine to twelve experiments. *: p<0.05versus control (vehicle). None of the antibodies affected baselineendothelial proliferation in the absence of VEGF (not shown). Ab:polyclonal antiserum; MoAb: monoclonal antibodies; bFGF: basicfibroblast growth factor.

5. PIGF Specifically Modulates the Responsiveness to VEGF

Since VEGF plays a role in the above-mentioned phenotypes ofangiogenesis, arteriogenesis and permeability, we investigated whetherPIGF determined the responsiveness to VEGF. Subcutaneous implantation ofmatrigel (A. Passaniti et al., 1992, Lab. Invest. 67, 519-528)supplemented with VEGF₁₆₅ (VEGF₁₆₅) induced a strong angiogenic responsein PIGF^(+/+) but not in PIGF^(−/−) mice (hemoglobin content: 0.28±0.02g/dl in PIGF^(+/+) mice versus 0.02±0.02 g/dl in PIGF^(−/−) mice; n=15;p<0.05).

In contrast, basic fibroblast growth factor (bFGF) induced a similarangiogenic response in both genotypes (hemoglobin content: 0.28±0.02g/dl in PIGF^(+/+) mice versus 0.25±0.02 g/dl in PIGF^(−/−) mice; n=15;p=NS). These observations were confirmed by histological analysis andimmunostaining for endothelial factor VIII-related antigen.

The reduced response of PIGF^(−/−) endothelial cells to VEGF wasconfirmed using cultured primary PIGF^(−/−) endothelial cells. VEGF₁₆₅(100 ng/ml) was chemotactic for PIGF^(+/+) but not for PIGF^(−/−)endothelial cells, whereas both genotypes responded comparably to bFGF.PIGF (100 ng/ml) itself was not chemotactic for endothelial cells ofeither genotype and did not affect the response of PIGF^(+/+)endothelial cells to VEGF, likely because endothelial cells alreadyproduce abundant PIGF. However, PIGF completely restored the impairedmigration of PIGF^(−/−) endothelial cells in response to VEGF₁₆₅. PIGFalso enhanced the chemo-attractive activity of VEGF on smooth musclecells, known to express VEGFR-1 and VEGFR-2 (C. L. Grosskreutz et al.,1999, Microvasc. Res. 58, 128-136). VEGF stimulated the migration ofPIGF^(+/+) but not of PIGF^(−/−) smooth muscle cells, whereas bFGFstimulated smooth muscle cells of both genotypes. Similar effects wereobserved for SMC proliferation. Even though PIGF alone did not stimulatethe cells, it rescued the impaired smooth muscle cell response to VEGF,further underscoring that PIGF is essential for the biological activityof VEGF. Thus, PIGF determines and synergistically amplifies theresponse to VEGF.

6. Inhibition of PIGF Impairs Pulmonary Hypertension

Wild-type mice are injected with different concentrations of a murineanti-PIGF antibody (0 μg, 1 μg, 5 μg, 10 μg and 50 μg). Murine anti-PIGFantibody is generated in the PIGF^(−/−) mouse. After 72 hours, the miceare placed for four weeks in a tightly sealed chamber under normobarichypoxia (FiO₂ 10%). After 28 days, the mice are sacrificed and used forhistological analysis as described in the materials and methods section.The control mouse with 0 μg anti-PIGF antibody developed a serioushypoxia-induced pulmonary vascular remodeling. The murine anti-PIGFantibody prevents this pulmonary vascular remodeling at very lowconcentrations.

7. Inhibition of PIGF Impairs Inflammation

Occlusion of a supply artery is a frequent complication ofatherosclerosis or arterial restenosis and deprives downstream tissuesof oxygen and nutrients. However, coincident enlargement of preexistingcollaterals due to endothelial activation and smooth muscle growth(adaptive arteriogenesis) may allow residual perfusion to the ischemicregions and prevent tissue necrosis in the territory of the occludedartery.

Even though administration of VEGF protein or VEGF gene transfer hasbeen shown to improve collateral growth, the role of endogenous VEGFremains controversial. PLGF has not been previously implicated in thisprocess. Macrophages play a role in adaptive arteriogenesis, but therole of PIGF remains unknown. Therefore, the role of macrophages inadaptive arteriogenesis of collateral arterioles was studied afterligation of the femoral artery. Mac3-positive macrophages, known to playan essential role in collateral growth, were found to adhere to theendothelium and to infiltrate in and through the wall of the collateralsthree days after ligation. However, more collaterals were infiltrated byMac3-positive macrophages in PLGF^(+/+) than in PLGF^(−/−) mice (45 of66 PLGF^(+/+) collaterals versus 29 of 67 PLGF^(−/−) collaterals; p<0.05by Chi-square analysis; n=5 mice). This may relate to the known monocytechemo-attractant activity of PLGF. Indeed, using another model ofleukocyte attraction (local endotoxin injection in the footpad),three-fold more leukocytes infiltrated in PLGF^(+/+) than in PLGF^(−/−)vessels (CD45-positive cells/vessel: 5.2±1 in PLGF^(+/+) mice versus1.5±0.2 μm in PLGF^(−/−) mice; n=5; p<0.05). Macrophages may alsomodulate collateral growth via production of PLGF (8±2 PLGF/10³ HPRTmRNA molecules; n=5). Another characteristic feature of adaptivearteriogenesis is the extravasation of fibronectin, providing a scaffoldfor migrating smooth muscle cells. Extravasation of fibronectin wasgreater in PLGF^(+/+) than in PLGF^(−/−) collaterals, as revealed by themore numerous collateral vessels, surrounded byfibronectin-immunoreactive deposits (57 of 80 PLGF^(+/+) collateralsversus 21 of 83 PLGF^(−/−) collaterals; n=5 mice; p<0.05 by Chi-Square).The increased permeability in PLGF^(+/+) collaterals may be caused bythe synergism between PLGF and VEGF, known to be released by activatedmacrophages. VEGF levels in thioglycolate-stimulated peritonealmacrophages were 200±11 VEGF/10³ HPRT mRNA molecules (n=5). Thus, PLGFis essential for collateral growth.

8. Preparation of Monoclonal Antibodies Against PIGF

Since PIGF deficiency reduces the phenotype of diverse pathologicalprocesses of angiogensis, arteriogenesis and vascular leakage comprisingischemic retinopathy, tumor formation, pulmonary hypertension, vascularleakage (edema formation) and inflammatory disorders, molecules that caninhibit the formation of PIGF, or binding PIGF to its receptor(VEGFR-1), or signal transduction initiated by PIGF, can be useful totreat the above-mentioned pathological processes. Monoclonal antibodiesagainst murine PIGF-2 were produced essentially as previously described(P. J. Declerck et al. (1995) J. of Biol. Chem. 270, 8397), however,using mice with inactivated PIGF genes. The mice were immunized bysubcutaneous injection of murine PIGF-2 (R&D Systems). In total, 120hybridomas were produced, of which 15 showed a 50% inhibition, 38 showed70% inhibition and five gave complete inhibition of binding of rmPIGF-2to its receptor (Flt-1). This was measured in an immunofunctional ELISAin which 96-well plates were coated with 100 μl of 1 μg/ml of rmFlt-1/Fcchimera overnight at room temperature in PBS. After blocking for onehour with 1% BSA in PBS, 100 μl of a mixture of 70 μl of hybridomamedium pre-incubated with 70 μl of recombinant mPIGF-2 at 10 ng/ml fortwo hours at room temperature was applied to the plate. A standard ofrmPIGF-2 ranging from 20 ng/ml to 156 pg/ml was included (diluted inPBS-Tween.BSA-EDTA). Plates were incubated one hour at 37° C. and onehour at room temperature, washed five times with PBS-Tween and 100 μl ofbiotinylated goat anti-murine PIGF-2 at 200 ng/ml was applied for twohours at room temperature. After washing five times with PBS-Tween, 100μl of avidin-HRP conjugate (Vectastorin ABC kit) was applied for onehour at room temperature. After washing five times with PBS-Tween, theplate was developed with 90 μl of o-phenylene diamine in citratephosphate buffer, pH 5.0, for 30 minutes and measured at 490 nm.

The five positive clones (PL1H5, PL5D11, PL9F7, PL13F11, PL17A10) weresubcloned, grown and injected in mice (PIGF knockouts in Balb/cbackground) to produce ascites. The monoclonal antibodies were purifiedon protein-A Sepharose and again tested for inhibition of binding ofm-PIGF-2 to Flt-1/Fc. The results (Table 3) indicate that Mab-PL5D11markedly inhibits binding of m-PIGF-2 to its receptor. This Mab wasselected for evaluation in the edema model (mustard oil skinapplication).

TABLE 3 Inhibition by anti-murine PIGF-2 Mab of murine PIGF-2 binding tomurine Flt-1/Fc. The data represent residual m-PIGF-2 in percent. Molarexcess versus m-PIGF-2 Nr 10 X 5 X 2.5 X 1.25 X No antibody 1 PL1H5G5 6664 63 89 100 2 PL5D11D4 10 15 22 43 100 PL5D11F10 14 19 22 35 100 3PL13F11C8 57 70 83 100 100 4 PL17A10E12 40 46 60 89 100 PL17A10F12 41 4153 90 100 Negative control Irrelevant antibody 1 C 8 100 100 100 100 100Concentration of m-PIGF-2 in ng/ml 5 5 5 5 5 Concentration of antibodyin ng/ml 200 100 50 25 0

9. Validation of the PIGF Monoclonals in a Mustard Oil Skin ApplicationModel

Mustard oil was painted on the ears of Swiss mice, and extravasation ofEvans blue was determined. Antibodies were injected intravenously at 300μg/kg 30 minutes before injection of Evans blue and application ofmustard oil. Briefly, 100 μl of a test agent was injected via a jugularvein catheter, followed 30 minutes later by an intravenous injection of300 μl of 0.5% Evans blue. One ear was painted with 0.1% mustard oil andrepainted again 15 minutes later. After 30 minutes, the mouse wasperfused via the left ventricle with saline containing 100 U/ml heparin,followed by 3% paraformaldehyde in citrate buffer. The ears wereamputated, dissected in small segments and extracted overnight informamide at 55° C. The absorbance of the extraction fluid was measuredat 610 nm.

Anti-PIGF antibodies, which blocked the PIGF response of endothelialcells (Table 4), reduced vascular leakage in wild-type mice applicationof mustard oil on their ears (relative absorbance units/ear: 13±3 aftermineral oil; 53±7 after mustard oil plus control IgGs; 26±5 aftermustard oil plus anti-PIGF; n=5; p<0.05).

TABLE 4 Effect of mustard oil on Evans blue extravasation. Group A⁶¹⁰ nmp No antibody 0.76 ± 0.09 — PL5D11D4 10 μg 0.50 ± 0.05 0.12 PL17A10E1210 μg 0.37 ± 0.04 0.03

10. Isolation of PIGF Inhibitors by Screening of a Tetrameric Library

A tetrameric library has been assayed for its ability to inhibit inELISA assays the binding between the recombinant mouse Placental GrowthFactor 2 (mPIGF-2) (R&D Systems Cat. No. 645-PL) and the recombinantmouse Vascular Endothelial Growth Factor Receptor 1 (VEGF R1) Flt-1/Fcchimera (R&D Systems, Cat. No. 471-F1). The ELISA assay was performedfollowing this procedure. The receptor was coated on a microtiter plate(Costar, Cat. No. 3590) at 1 μg/ml in NaH₂PO₄ 50 mM, NaCl 150 mM pH 7.5(PBS), 100 μl/well, for 16 hours at room temperature. The wells werewashed five times with PBS containing 0.004% Tween (PBS-T) and blockedwith Bovine Serum Albumin (BSA) 1% in PBS, 150 μl/well, for two hours atroom temperature. The plate was washed with PBS-T five times, and 100 μlof mPIGF diluted at 8 ng/ml in PBS pH 7.5, BSA 0.1%, EDTA 5 mM, Tween0.004% (PBET), was added to each well. After the incubation for one hourat 37° C. and one hour at room temperature, the plate was washed again,and biotinylated anti-mPIGF antibodies (R&D Systems, Cat. No. BAF 465)were added at 200 ng/ml in PBET, 100 μl/well, and incubated for twohours at room temperature. The plate was washed five times with PBS-T,100 μl/well of a solution containing an avidin-biotin system(Vectastatin Elite ABC kit, Vector Laboratories, Cat. No. PK 6100). Thissolution was prepared by mixing one drop of solution A and one drop ofsolution B in 2.5 ml of Tris-HCl 50 mM and diluting this mix 1/100 inPBET. After one hour of incubation, the plate was washed as above, and90 μl/well of a solution containing 1 mg/ml of O-Phenylenediamine (OPD)in citrate buffer pH 5.0 was added to each well. After 40 minutes, thedeveloping reaction was stopped by adding 30 μl/well of sulfuric acid3M. The plate was read at 490 nm with an ELISA reader. To test theinhibitory activity of the library, each pool of the library was addedin competition with mPIGF using a molar excess of 1000 times (1:1000,1:2000 means a molar excess of 2000; 1:1500 means a molar excess of1500, etc.). The results of the screening are shown in Table 5.

TABLE 5 plate 1 OD 490 nm mPIGF 8 ng/ml 100 0.993 mPIGF 5 ng/ml67.673716 0.672 R&D mAb (1:5) 51.0574018 0.507  1 D-Ala 80.5639476 0.8 2 D-Asp 75.5287009 0.75  3 D-Val 80.5639476 0.8  4 D-Glu 57.40181270.57  5 L-Cha 87.6132931 0.87  6 D-Phe 79.0533736 0.785  7 D-Thr83.5850957 0.83  8 D-Met 84.592145 0.84  9 D-Cys(Acm) 91.6414904 0.91 10D-Lys 88.8217523 0.882 11 D-Tyr 90.6344411 0.9 12 D-Pro 93.6555891 0.9313 D-Leu 95.367573 0.947 14 D-His 107.75428 1.07 15 D-Gln 101.7119841.01 16 D-Trp 101.711984 1.01 17 D-Arg 105.740181 1.05 18 D-Asn106.747231 1.06 19 D-Ile 103.222558 1.025 20 D-Arg(Tos) 103.323263 1.026plate 2 OD 490 nm mPIGF 8 ng/ml 100 0.991 mPIGF 5 ng/ml 71.6448032 0.7121 D-Ser 83.6528759 0.829 22 L-Cys(Acm) 83.8546922 0.831 23 L-Cys(Bzl)84.7628658 0.84 24 L-Cys(p-MeBzl) 89.3037336 0.885 25 L-Cys(tBu)92.0282543 0.912 26 L-Mt(O) 83.9556004 0.832 27 L-Met(O2) 78.60746720.779 28 L-Glu(a-OaII) 92.244898 0.904 29 b-Ala 100 0.98 30 Gly92.1428571 0.903  4 D-Glu 1:500 86.1884368 0.805  4 D-Glu 1:100059.1006424 0.552  4 D-Glu 1:1500 48.2869379 0.451  4 D-Glu 1:200039.9357602 0.373  2 D-Asp 1:500 89.9357602 0.84  2 D-Asp 1:100078.9079229 0.737  2 D-Asp 1:1500 78.0513919 0.729  2 D-Asp 1:200074.9464668 0.7 14 D-His 1:500 96.6809422 0.903 14 D-His 1:1000101.391863 0.947 14 D-His 1:1500 100.535332 0.939 14 D-His 1:200096.8950749 0.905

Thirty pools of 900 different tetrameric peptides were screened for thepossibility to interfere with the binding of mPIGF-2 to mVEGFR-1.Clearly, pool 4 on Table 5 (with D-Glu on the terminal position) hasinhibitory activity when compared with two anti-PIGF antibodies tointerfere with binding (R&D mAb and a homemade mAb) of mPIGF-2 tomVEGFR-1.

Materials and Methods Models of Angiogenesis

Morphometric analysis of myocardial, renal, and retinal angiogenesis inneonatal mice was performed as described (P. Carmeliet et al., 1999,Nat. Med. 5, 495-502).

Matrigel assay: Ingrowth of capillaries in matrigel was performed asdescribed (A. Passaniti et al., 1992, Lab. Invest. 67, 519-528).Briefly, 500 μl ice-cold matrigel containing heparin (300 μg/ml) andVEGF (100 ng/ml), or basic fibroblast growth factor (bFGF; 100 ng/ml)was injected subcutaneously. After seven days, the matrigel pellet withthe neovessels was dissected for analysis of neovascularization: onepart was homogenized to determine the hemoglobin content determinedusing Drabkin's reagent (Sigma, St. Quentin Fallavier, France), whereasthe other part was fixed in 1% paraformaldehyde for histologicalanalysis.

ES-tumor model: For ES cell-derived tumor formation, 5×10⁶ ES cells weresubcutaneously injected into PIGF^(+/+) Nu/Nu or PIGF^(−/−) Nu/Nu mice,obtained by intercrossing PIGF^(+/−) Nu/Nu mice, as described (P.Carmeliet et al., 1998, Nature 394, 485-490). Vascular densities werequantitated by counting the number of endothelial cords and capillaries(diameter <8 μm), medium-sized vessels (diameter 10-25 μm), or largevessels (diameter >30 μm) per field (1.2 mm²) in six to eight randomlychosen optical fields on three to five adjacent sections (320 μm apart)per tumor using the Quantimet Q600 imaging system.

Ischemic retina model: Retinal ischemia was induced by placing P7neonatal mice in a cage of hyperbaric (80%) oxygen for five days, afterwhich they were returned for another five days in normal room air, asdescribed (L. E. Smith et al., 1994, Invest. Opthalmol. Vis. Sci. 35,101-111). Fluorescent retinal angiography and endothelial cell counts onretinal cross-sections were determined as reported (H. P. Hammes et al.,1996, Nat. Med. 2, 529-533). Venous dilatation and arterial tortuositywere semi-quantitatively scored on a scale from 0-3.

Wound models: Vascular remodeling during skin wound healing was analyzedwithin four days after occurrence of a 10 mm full-thickness skin woundon the back of the mouse, as described (S. Frank et al., 1995, J. Biol.Chem. 270, 12607-12613). Wound healing was scored by daily measurementsof the width of the wound.

Pulmonary hypertension: Adult mice were placed for four weeks in atightly sealed chamber under normobaric hypoxia (FiO₂ 10%), as described(C. A. Hales et al., 1983, Am. Rev. Respir. Dis. 128, 747-751). After 28days, mice were used for measurements of hematocrit, using an automatedcell counter (Abbott Cell-Dyn 1330 system, Abbott Park, Ill.) and forhistological analysis. Right ventricular pressures (RVP) were measuredin anesthetized ventilated mice (sodium pentobarbital, 60 mg/kg, i.p.)by transthoracic puncture using high-fidelity pressure micromanometers(Millar) after inhalation of a gas mixture containing 20% O₂ or 7% O₂.For histology, mice were perfused with 1% phosphate-bufferedparaformaldehyde at 100 cm H₂O pressure via the heart and at 30 cm H₂Opressure through the trachea. Visualization of the internal elasticlamina (EEL) and external elastic lamina (EEL) was achieved using Hart'selastin stain. Hypoxia-induced pulmonary vascular remodeling wasassessed by counting the number of non-muscularized (only IEL) andpartially (IEL plus incomplete EEL) or fully (IEL plus complete EEL)muscularized peripheral vessels (landmarked to airway structures distalto the terminal bronchioli) per 100 alveoli in fields containing 5×500alveoli (C. A. Hales et al., 1983, Am. Rev. Respir. Dis. 128, 747-751).

Vascular Permeability

Arthus reaction (allergen-induced edema formation in the skin (J.Casals-Stenzel et al., 1987, Immunopharmacology 13, 177-183)): mice weresensitized by intraperitoneal (i.p.) injection of saline (1 ml/kg)containing ovalbumin (40 μg/kg; Sigma, St. Louis, Mo.) and Al(OH)₃ (0.2mg/ml added to the antigen solution one hour prior to injection) on days0 and 2.

Vascular leakage was quantified 14 days after presensitization bydetermining the amount of intravenously injected 125I-bovine serumalbumin (BSA) and Evans blue dye accumulating in the skin injectionsite. Therefore, the fur on the dorsal skin of anaesthetized mice wasshaved, and 1.5 μCi/kg of 125I-BSA (2.8 μCi/μg; NEN-Dupont, France)mixed with a solution of Evans blue dye in sterile saline (15 mg/kg) wasinjected i.v. Ten minutes later, ovalbumin (100 ng/site) was injected atfour intradermal sites. After 60 minutes, the degree of vascular leakagewas quantified: (i) by measuring the diameter of the edematous spot(visualized by its coloration) using a micrometer; and (ii) bydetermining the amount of extravasated plasma protein at each skin site(expressed as μl extravasated plasma) after normalizing the 125I-cpm inthe skin (10 mm punch) for the 125I-cpm in 1 μl of plasma.

Miles assay: Vascular permeability was assayed using the Miles assay (N.McClure, 1994, J. Pharmacol. Toxicol. Methods). Briefly, mice wereshaved and injected with 50 μl of a solution containing 0.5% Evans bluein saline 45 minutes prior to intradermal injection of 20 μlphosphate-buffered saline (PBS) containing 1, 3 or 10 ng recombinanthuman VEGF₁₆₅; pictures were taken 45 minutes later.

Skin healing: After shaving, a standardized 15 mm full-thickness skinincision was made on the back of anesthetized mice, taking care not todamage the underlying muscle. Extravasation of 125I-BSA (expressed as gplasma/g tissue/min) was measured as described (R. G. Tilton et al.,1999, Invest. Opthalmol. Vis. Sci. 40, 689-696).

In Vitro Angioaenesis Assays

Endothelial and smooth muscle cell culture: In order to obtain mousecapillary endothelial cells, anesthetized mice were injected s.c. with500 μl of ice-cold matrigel containing bFGF (100 ng/ml) and heparin (100μg/ml). After seven days, the matrigel pellet was dissected andenzymatically dispersed using 0.1% type II collagenase (Sigma, St.Louis, Mo.). Mouse endothelial cells were routinely cultured in T75flasks coated with 0.1% gelatin in M131 medium supplemented with 5% MVGS(Gibco-BRL). Smooth muscle cells from mouse aorta were harvested andcultured as described (J. M. Herbert et al., 1997, FEBS Lett. 413,401-404). Before stimulation, cells were starved in medium with 0.5%serum for 24 hours, after which they were stimulated with human VEGF₁₆₅and/or murine PIGF, or bFGF (all from R&D, Abingdon, UK) for 24 hoursbefore analysis of the total cell number (proliferation) or the numberof cells migrated after scrape-wounding (migration).

Synthesis of a Tetrameric Library

A tetrameric library has been synthesized using commercially available9-Fluorenylmethoxycarbonyl (Fmoc)-derivatized amino acids (purity >99%).All derivatives are listed in Table 6 along with catalog numbers andcompany names (provider) from which they have been purchased.

TABLE 6 List of building blocks used throughout the peptide librarypreparation. Building block Number 3-letter code Building blockProtected derivative Provider Catalog number 1 D-Ala D-AlanineNα-Fmoc-D-Alanine Chem-Impex Intl 02372 2 D-Asp D-Aspartic acidNα-Fmoc-D-Aspartic acid (t-butyl Chem-Impex Intl 02478 ester) 3 D-ValD-Valine Nα-FmocD-Valine Chem-Impex Intl 02471 4 D-Glu D-Glutamic acidNα-Fmoc-D-Glutamic acid (t-butyl Chem-Impex Intl 02479 ester) 5 L-ChaL-Cyclohexylalanine Nα-Fmoc-L-Cyclohexylalanine Sygena FC-01-003-117 6D-Phe D-Phenylalanine Nα-Fmoc-D-Phenylalanine Novabiochem 04-13-1030 7D-Thr D-Threonine Nα-Fmoc-D-Threonine Chem-Impex Intl 02483(O-t-butyl-ether) 8 D-Met D-Methionine Nα-Fmoc-D-Methionine Novabiochem04-13-1003 9 D-Cys(Acm) D-Cysteine (S- Nα-Fmoc-D-Cysteine (S-Novabiochem 04-13-1054 acetamydomethyl) acetamydomethyl) 10 D-LysD-Lysine Nα-Fmoc-D-Lysine (N^(ε)-t- Alexis 104-041-G005butyloxycarbonyl) 11 D-Tyr D-Tyrosine Nα-Fmoc-D-Tyrosine(O-t-butyl-ether) Chem-Impex Intl 02465 12 D-Pro D-ProlineNα-Fmoc-D-Proline Novabiochem 04-13-1031 13 D-Leu D-LeucineNα-Fmoc-D-Leucine Chem-Impex Intl 02427 14 D-His D-HistidineNα-Fmoc-D-Histidine (N^(r)-trytil) Alexis 104-034-G005 15 D-GlnD-Glutamine Nα-Fmoc-D-Glutamine (N-trytil) Novabiochem 04-13-1056 16D-Trp D-Triptophan Nα-Fmoc-D-Triptophan (N^(in)-t- Chem-Impex Intl 02484butyloxycarbonyl) 17 D-Arg D-Arginine Nα-Fmoc-D-Arginine (N^(r)- Alexis104-113-G005 pentamethylchroman) 18 D-Asn D-AsparagineNα-Fmoc-D-Asparaqine (N-trytil) Chem-Impex Intl 02477 19 D-IleD-Isoleucine Nα-Fmoc-D-Isoleucine Chem-Impex Intl 03448 20 D-Arg(Tos)D-Arginine (N^(r)-Tosyl) Nα-Fmoc-D-Arginine (N^(r)-Tosyl) Chem-ImpexIntl 02382 21 D-Ser D-serine Nα-Fmoc-D-serine (O-t-butyl-ether) Alexis104-066-G005 22 L-Cys(Acm) L-Cysteine (S- Nα-Fmoc-L-Cysteine (S-Chem-Impex Intl 02396 acetamydomethyl) acetamydomethyl) 23 L-Cys(Bzl)L-Cysteine (S-benzyl) Nα-Fmoc-L-Cysteine (S-benzyl) Novabiochem04-12-1015 24 L-Cys(p- L-Cysteine (S-p-methyl- Nα-Fmoc-L-Cysteine(S-p-methoxy- Chem-Impex Intl 02399 MeBzl) benzyl) benzyl) 25 L-Cys(tBu)L-Cysteine (S-tert-butyl) Nα-Fmoc-L-Cysteine (S-tert-butyl) Novabiochem04-12-1016 26 L-Met(O) L-Methionine-sulphoneNα-Fmoc-L-Methionine-sulphone Novabiochem 04-12-1112 27 L-Met(O)₂L-Methionine-sulphoxide Nα-Fmoc-L-Methionine-sulphoxide Novabiochem04-12-1113 28 L-Glu(β-OAll) L-Glutamic acid-(β-allyl) Nα-Fmoc-L-Glutamicacid-(β-allyl) Novabiochem 04-12-1158 29 β-Ala β-AlanineNα-Fmoc-β-Alanine Chem-Impex Intl 02374 30 Gly Glycine Nα-Fmoc-GlycineChem-Impex Intl 02416

The library has been built onto the following scaffold (G. Fassina etal. (1996) J. Mol. Recogn. 9, 564; M. Marino et al. Nat. Biotechn.(1997) 18, 735):

where G represents the amino acid Glycine and K represents the aminoacid L-Lysine onto which three levels of randomization have beenachieved by applying the Portioning-Mixing method (A. Furka et al.(1991) Int. J. Pept. Protein. Res. 37, 487; K. S. Lam (1991) Nature 354,82). The total number of peptides generated (N_(t)) can be calculated bythe following formula:

N_(t)=B^(x)

Where B is the number of building blocks used (30) and x is the numberof randomization (3).

a) Synthesis of the Scaffold

The initial scaffold has been prepared by manual solid phase synthesisstarting from 419 mg of Fmoc-Gly-derivatized4-hydroxymethylphenoxyacetic polystyrene resins (PS-HMP) (substitution0.75 mmol/g, Novabiochem Cat. No. 04-12-2053), corresponding to 314 μmolof Glycine onto which two subsequent couplings with Fmoc-L-Lys(Fmoc)-OH(Chem-Impex Intl. Cat. No. 01578) have been carried out. The resin hasbeen placed in a 35 ml polypropylene cartridge endowed with apolypropylene septum (AllTech, Cat. No. 210425) and washed three timeswith 4.0 ml of N,N-dimethylformamide (DMF, Peptide synthesis grade,LabScan, Cat. No. H6533). To remove the Fmoc protection, the resin hasbeen treated for 15 minutes with 5.0 ml of 20% Piperidine (BIOSOLVE LTD,Cat. No. 16183301) in DMF and then washed three times with 4.0 ml ofDMF. For the coupling of the first Lysine, 1.5 mmol ofFmoc-L-Lys(Fmoc)-OH (0.87 g) have been dissolved in 6.0 ml of DMF andthen activated by adding 3.0 ml of a 0.5 M solution of2-(1H-Benzotriazol-yl)-1,1,3,3-tetramethyl-uronium tetrafluoroborate(TBTU, >99%, Chem-Impex Intl, Cat. No. 02056) and 1-Hydroxybenzotriazole(HOBt, SIGMA-ALDRICH, Cat. No. H2006) in DMF and 3.0 ml of a 1 Msolution of Di-isopropyl-ethylamine (DIEA, SIGMA-ALDRICH, Cat. No.D-3387) in DMF. After four minutes stirring at room temperature, thesolution has been transferred onto the resin and stirred for 30 minutes.To remove the excess of amino acid, the resin has been washed threetimes with 4.0 ml of DMF. The deprotection of the Lysine-Fmoc groups hasbeen achieved by treating the resin for 15 minutes with 5.0 ml of 20%Piperidine in DMF and washing three times with 4.0 ml of DMF to removethe excess of reagent. For the coupling of the second Lysine, 3.0 mmolof Fmoc-L-Lys(Fmoc)-OH (1.77 g) have been dissolved in 6.0 ml of DMF andthen activated adding 6.0 ml of a 0.5 M solution of TBTU/HOBt in DMF and6.0 ml of DIEA in DMF. After four minutes stirring at room temperature,the solution has been transferred onto the resin and stirred for 30minutes. The resin has then been washed three times with 4.0 ml of DMF.The final Fmoc groups have been removed by treatment with 10.0 ml of 20%Piperidine in DMF for 15 minutes. The resin has then been submitted tothe following washings:

Number of Solvent washes Volume (ml) DMF 3 4.0 MeOH* 3 4.0 Et₂O** 3 4.0*Methanol (MeOH, LabScan, Cat. No. A3513) **Ethyl Ether (Et₂O, LabScan,Cat. No. A3509E)

The resin has been dried by applying a Nitrogen stream and then weighed.The final weight of the resin was 442 mg.

b) Assembly of the Library

The library has been generated by applying the following procedure:

b.1 Resin Splitting into 30 Equal Aliquots.

The resin has been transferred into a 50 ml polypropylene graduated tubeand 35 ml of DMF:DCM (2:3) (DCM, Dichloromethane, LabScan, Cat. No.H6508L) have been added. The suspension has been thoroughly mixed andfractions of 1.0 ml have been dispensed in 30 polypropylene syringes (3ml) endowed with filtration septa at the bottom (Shimadzu Corp. Cat. No.292-05250-02). The remaining volume of suspension has been diluted up to35 ml with DMF:DCM (2:3) and 1.0 ml fractions have been again dispensedinto the syringes. The graduated tube has been once again filled up to30 ml and 1.0 ml aliquots distributed into the syringes. The syringeshave been vacuum drained from the bottom and the resins washed once with1.0 ml of DMF. The syringes, labeled with numbers from 1 to 30,contained an equal fraction of resin corresponding to around 10 μmolesof scaffold (40 μmoles of NH₂ groups).

b.2 Coupling of the First Random Residue (Position 3).

0.75 M stock solutions in DMF of the 30 amino acids (listed in Table 5)have been prepared and stored at 4° C. until use. To carry out couplingsand deprotection on sub-libraries, from this step forward, a PSSM8eight-channel peptide synthesizer (Shimadzu Corp.) has been used. Thesyringes have been placed into the synthesizer and 267 μl (200 μmoles)of amino acids have been dispensed into 2 ml polypropylene tubes(Eppendorf, Cat. No. 24299). The machine automatically performsactivations, acylations, deprotections and washings. Activations werecarried out using 400 μl of a 0.5 M solution in DMF of TBTU/HOBt plus400 μl of a 1 M solution in DMF of DIEA. Acylations have been carriedout for 30 minutes, mixing the suspensions by bubbling nitrogen from thebottom of the syringes. Deprotection have been carried out for 15minutes with 0.9 ml of 20% piperidine/DMF, while washings have beenperformed with 0.9 ml of DMF (three times, two minutes).

In the first round, amino acids 1 to 8 have been coupled, in the secondround, amino acids 9 to 16 were coupled, in the third round, amino acids17 to 24 were coupled, and in the final round, amino acids 25 to 30 werecoupled.

b.3 Mixing and Re-Splitting of Resins.

To each syringe, 500 μl of DMF:DCM (2:3) have been added and the resinssuspended by gentle swirling. The suspensions have been removed from thesyringes, collected in a 50 ml polypropylene tube (graduated) andthoroughly mixed by vigorous shaking.

After addition of DMF:DCM (2:3) up to a final volume of around 35 ml,1.0 ml aliquots of suspension have been re-dispensed into the 30syringes, repeating the operations described in step 1 (“Resin splittinginto 30 equal aliquots”). At the end, the resins have been washed oncewith 1.0 ml of DMF.

b.4 Coupling of the Second Random Residue (Position 2).

All operations described in step 2 (“Coupling of the first randomresidue”) were repeated.

b.5 Mixing and Re-Splitting of Resins.

All operations described in step 3 (“Mixing and re-splitting of resins”)were repeated.

b.6 Coupling of the Third Known Residue (Position 1).

All operations described in step 2 (“Coupling of the first randomresidue”) have been repeated.

b.7 Final Washes and Drying of the Resins.

The resins were washed three times with 1 ml of DCM, three times with 1ml of MeOH, and two times with 1 ml of Et₂O. The resins have then beendried under vacuum.

b.8 Cleavage.

Thirty ml of a TFA-H₂O-TIS (100:5:5, v/v/v) mixture (TIS,tri-iso-propylsilane, SIGMA-ALDRICH Cat. No. 23, 378-1) have beenfreshly prepared and 800 μl added to each syringe. After vortexing forthree hours, the resins have been filtered off, collecting the acidicsolution directly in 15 ml polypropylene tubes (labeled with numbersfrom 1 to 30) containing 5 ml of cold Et₂O. The white precipitates havebeen separated by centrifugation at 3000 rpm for ten minutes and theorganic solvents discarded. The precipitates have been washed once with5 ml of cold Et₂O and, after centrifugation, were dissolved in 2.0 ml ofH₂O/CH₃CN/TFA 50:50:01 and lyophilized. Ten mg/ml stock solutions of thepeptide libraries in DMSO have been prepared and stored in sealed vialsat −80° C.

1. A method of suppressing inflammation in a mammal, comprisingadministering to a mammal in need of suppressing inflammation a moleculethat is capable of neutralizing the activity of placental growth factor,such that said inflammation is suppressed.
 2. The method according toclaim 1 wherein said inflammation is an inflammatory disorder orinflammatory disease.
 3. A method of suppressing development ofinflammation-associated edema in a mammal, comprising administering to amammal likely to develop inflammation-associated edema a molecule thatis capable of neutralizing the activity of placental growth factor, suchthat development of inflammation-associated edema is suppressed.
 4. Themethod according to claim 1 wherein said molecule is an antibody or afragment thereof capable of neutralizing the activity of placentalgrowth factor.
 5. The method according to claim 4 wherein said antibodyis a monoclonal antibody.
 6. The method according to claims 4 or 5wherein said antibody is a human or humanized antibody.
 7. The methodaccording to claim 4 wherein said antibody fragment is a Fab, F(ab)′2 orscFv fragment.
 8. A method of reducing inflammation in a mammal,comprising administering to a mammal suffering from inflammation amolecule that inhibits the activity of placental growth factor, suchthat said inflammation is reduced.
 9. The method according to claim 8wherein said inflammation is an inflammatory disorder or inflammatorydisease.
 10. A method of reducing development of inflammation-associatededema in a mammal, comprising administering to a mammal likely todevelop inflammation-associated edema a molecule that inhibits theactivity of placental growth factor, such that development ofinflammation-associated edema is reduced.
 11. The method according toclaim 8 wherein said molecule is an antibody or a fragment thereofcapable of inhibits the activity of placental growth factor.
 12. Themethod according to claim 11 wherein said antibody is a monoclonalantibody.
 13. The method according to claims 11 or 12 wherein saidantibody is a human or humanized antibody.
 14. The method according toclaim 11 wherein said antibody fragment is a Fab, F(ab)′2 or scFvfragment.